Stephan Niland (100766.3150 at compuserve.com) <100766.3150 at compuserve.com>
wrote on the subject: cell cycle synchronization
>Hi, out there,
>I ve got three questions on cell culture synchronization:
>1)how can I best synchronize a fibroblast cell culture?
>Is mitotic shake-off really the best, or are there any
>protocols using certain drugs? If yes, please mail me
>the respective reference or protocol!
Stephan,
This depends on what you want to do.
Mitotic shake off is good for studying mitotic and early G1 events in
a small population of cells, because it can give you over 90% of the
cells in mitosis. But if you need to do biochemistry on a
large number of cells, then you need a method that synchronizes the
entire population. No method gives you completrely synchronous cells.
Nocodozole blocked and selected cells move thru mitosis quite
synchronously after removel of the Nocodazole (Zieve et al., Exp.
Cell Res. 126:397.)
>2) How long will the culture probably be synchronous?
Only for a short period of time. Most cells lose synchrony in G1
because it is the most variable phase, even in constant cell culture
conditions.
>3) Are there possibilities to arrest such cultures in G1-,
> S-, G2-, or M-phase? if yes, how can I do it?
Cell cycle researchers most commonly synchronize cells at the G1-S
boundary with reversible inhibition of DNA synthesis. One way is the
double excess-thymidine block, that has the advantage of being
non-toxic and very reversible. These cells stay fairly well
synchronized until the next G1, so are good for S, G2 and even M
phase events. Also one can select cells in partyicular phases with
FACS (automated cell sorter).
See Mitchison, The Biology of the Cell Cycle
Zeuthen, Synchrony in Cell Division and Growth
Ahearne, An intoduction to Cell Population Kinetics.
>I would be glad, if you could help me, and I am looking
>forward to receiving your mail!
>Stephan (e-mail: 100766.3150 at compuserve.com)
-Dennis
Dr. M. Dennis Goode Phone (301) 405-6917
Department of Zoology Fax (301) 314-9358
University of Maryland e-mail goode at zool.umd.edu
College Park MD 20742
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"If the Lord Almighty had consulted me before embarking upon the
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